Depolarization of a skeletal muscle fiber causes calcium ions to be released from the sarcoplasmic reticulum (SR), the internal calcium- sequestering membrane system in muscle. The resulting elevated myoplasmic [Ca2+] enables contractile activity by binding to thin filament troponin C, which removes the inhibition of interaction between the thick and thin filaments. Elevated [Ca2+] also activates a variety of other [Ca2+] dependent regulatory changes in the fiber. On repolarization calcium release stops, [Ca2+] declines, calcium dissociates from troponin C and the muscle relaxes. The properties and functional roles of calcium binding proteins and the SR calcium pump in lowing [Ca2+] will be studied using two [Ca2+] indicators in voltage clamped single skeletal fibers from frog, rat and rabbit. The role of the soluble calcium and magnesium binding protein parvalbumin will be determined by comparing both the decline of [Ca2+] and the [Mg2+] transient in parvalbumin-containing and -lacking mammalian fast- and slow-twitch fibers. Modulation of the SR calcium pump in slow-twitch fibers by the SR protein phospholamban will be evaluated. The fast and slow phases of decline of [Ca2+] after various patterns of action potentials in each fiber type will be determine and the implications for [Ca2+] dependent regulation will be considered. The possible role of cytosolic [Ca2+] in altering fiber type during long-term electrical stimulation of tissue cultured fast- and slow-twitch fibers will be examined. Ca2+ indicators in the ER of cell lines transfected with various isoforms of the SR calcium pump or mutants thereof will be used to image [Ca2+] and changes in [Ca2+] within the internal stores to evaluate pump function in a simplified environment. [Ca2+] will also be monitored within the SR of muscle fibers to evaluate the roles of internal and external [Ca2+] in the function of the calcium pump in its normal environment. A variety of preparative, electrical, optical and computer analysis and modelling procedures previously developed and used in this laboratory will be combined with various tissue culture, molecular biological and digital imaging and image analysis techniques that are new to our lab. The results of these studies may provide basic information relevant to pathologic conditions and possible therapeutic interventions in various muscular, neuro-muscular or cardiac disease states.